Maintenance of the Shigella sonnei virulence plasmid is dependent on its repertoire and amino acid sequence of toxin:antitoxin systems

Shigella sonnei is a major cause of bacillary dysentery, and of increasing concern due to the spread of multi-drug resistance. S. sonnei harbours pINV, a ∼ 210 kb plasmid that encodes a Type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously in S. sonnei at high frequency, hampering studies on and vaccine development against this important pathogen. Here we investigated the molecular basis for the emergence of avirulence in S. sonnei , and show that avirulence mainly results from pINV loss, consistent with previous findings. Ancestral deletions have led to the loss from S. sonnei pINV of two toxin:antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV in Shigella flexneri . We show that introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contribute to pINV loss. Avirulence also results from deletions of T3SS-associated genes on pINV through recombination between insertion sequences (ISs) on the plasmid; these events differ from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrate that TA systems and ISs influence plasmid dynamics and loss in S. sonnei , and could be exploited for the design and evaluation of vaccines. IMPORTANCE Shigella sonnei is the major cause of shigellosis in high-income and industrialising countries, and an emerging multi-drug resistant pathogen. A significant challenge when studying this bacterium is that it spontaneously becomes avirulent during growth in the laboratory, through loss of its virulence plasmid (pINV). Here we decipher the mechanisms leading to avirulence in S. sonnei and how the limited repertoire and amino acid sequences of plasmid-encoded toxin:antitoxin (TA) systems make the maintenance of pINV in this bacterium less efficient compared with Shigella flexneri . Our findings highlight how subtle differences in plasmids in closely-related species have marked effects and could be exploited to reduce plasmid loss in S. sonnei . This should facilitate research on this bacterium and vaccine development.

Characterization of a New and Efficient Polyvalent Phage Infecting E. coli O157:H7, Salmonella spp., and Shigella sonnei

Ongoing outbreaks of foodborne diseases remain a significant public health concern. Lytic phages provide promising attributes as biocontrol agents. This study characterized KFS-EC3, a polyvalent and lytic phage, which was isolated from slaughterhouse sewage and purified by cesium chloride density centrifugation. Host range and efficiency of plating analyses revealed that KFS-EC3 is polyvalent and can efficiently infect E. coli O157:H7, Salmonella spp., and Shigella sonnei. KFS-EC3 had a latent time of 20 min and burst size of ~71 phages/infected cell. KFS-EC3 was stable and infectious following storage at a pH range of 3 to 11 and a temperature range of −70°C to 60°C. KFS-EC3 could inhibit E. coli O157:H7 growth by 2 logs up to 52 h even at the lowest MOI of 0.001. Genomic analysis of KFS-EC3 revealed that it consisted of 167,440 bp and 273 ORFs identified as functional genes, without any genes associated with antibiotic resistance, virulence, allergenicity, and lysogenicity. This phage was finally classified into the Tequatrovirus genus of the Myoviridae family. In conclusion, KFS-EC3 could simultaneously infect E. coli O157:H7, S. sonnei, and Salmonella spp. with the lowest MOI values over long periods, suggesting its suitability for simultaneous pathogen control in foods.

Evaluation of Lactic Acid Bacteria (LAB) against Shigella sonnei ATCC 25931

The main functions of Lactic Acid Bacteria (LAB) are the production of organic acids and their characteristic as inhibitors of pathogenic microorganisms. Disease caused by Shigella sonnei is a public health risk factor, as is the resistance they develop to antibiotics. Accordingly, in this study 4 LAB isolated from coffee, quesillo, and breast milk were evaluated under in vitro conditions on Shigella sonnei, considering the presence and cell-free filtration of LAB. The LAB strains were also studied via an antibiotic test using Penicillin, Ceftriaxone, and Ciprofloxacin. The results showed antagonistic activity against Shigella sonnei due to the presence of cells and organic acids. However, the LAB bacteriocins did not inhibit the pathogen. Furthermore, LABs were sensitive to the antibiotics tested. Products from the region such as coffee and quesillo, as well as breast milk are important for their nutritional value and for their LAB content. It would be useful to involve biotechnological research in the coffee and dairy chain on the one hand, and to promote breastfeeding on the other. The tests carried out are qualitative and the inhibitory effect is attributed to the strain and not to a generalization of species. It would also be useful to perform molecular identification of the lactic acid strains studied to introduce them in other food matrices, to evaluate their viability, and to carry out in vivo tests to assess their antimicrobial activity.

Avaliação das atividades antimicrobiana e citotóxica de frações ricas em alcaloides obtidas das partes aéreas de Mitracarpus frigidus (Rubiaceae)

O presente estudo teve como objetivo extrair e avaliar o potencial antimicrobiano e citotóxico de frações ricas em alcaloides obtidas a partir das partes aéreas de Mitracarpus frigidus. O isolamento de alcaloides foi realizado por meio de extração ácido-base e cromatografia em coluna com Sephadex LH-20 como fase estacionária. A atividade antimicrobiana foi avaliada pelo método de microdiluição em caldo para determinação da concentração inibitório mínima (CIM) e também por parâmetros quantitativos antimicrobianos (atividade total, percentual de atividade e índice de susceptibilidade microbiano) contra nove linhagens de microrganismos: Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, Klebsiella pneumoniae, Escherichia coli, Bacillus cereus, Candida albicans e Cryptococcus neoformans. A citotoxicidade foi testada frente a Artemia salina e duas linhagens de células leucêmicas, HL60 e Jurkart. A fração mais rica em alcaloides apresentou atividade expressiva para S. aureus, S. typhimurium, B. cereus, P. aeruginosa, S. sonnei e C. neoforman com CIM abaixo de 0,100 mg/mL. As amostras não apresentaram efeito citotóxico frente a A. salina e em relação as linhagens de células tumorais, somente as frações mais ricas em alcaloides apresentaram uma moderada atividade com cerca 50 e 35% de diminuição da viabilidade celular para HL60 e Jurkart, respectivamente. Os resultados apresentados abrem perspectivas para o futuro isolamento, purificação e identificação de substâncias bioativas de M. frigidus que possam ser utilizadas, principalmente no tratamento de infecções microbianas.

Antibiotic susceptibility of four Enterobacteriaceae strains (Enterobacter cloacae, Citrobacter freundii, Salmonella typhi and Shigella sonnei) isolated from wastewater, surface water and groundwater in the equatorial zone of Cameroon (Central Africa)

The health problems caused waterborne pathogens bacteria belonging to the Enterobacteriaceae family are aggravated by the rise of antimicrobial resistant bacteria phenomenon. This has been identified as one of the biggest global health challenges. The present study aims to make a comparative approach of the sensitivity/resistance to antibiotics of 4 Enterobacteriaceae Enterobacter cloacae, Citrobacter freundii, Salmonella typhi and Shigella sonnei isolated from wastewater, streams and groundwater in Yaounde town (Cameroon, Central Africa). Bacteria were isolated using standard methods their identification using the API 20E systems. The antibiogram tests were carried out using the Müller-Hinton antibiotic disc diffusion method. Results showed that inhibition diameters of antibiotic varied (P<0.05) with respect to the bacterial species on one hand, and with respect to the type of water harbored cells on the other hand. The proportion of bacterial strains of each isolated species, and which were sensitive or resistant varied from one antibiotic to another within the same antibiotics family. For β-lactams tested, no E. cloacae strain has been sensitive. The proportion of sensitive strains of other species varied between 5.88% and 23.53%. For the 2 aminoglycosides tested, 5.88% of the strains of each isolated species were resistant to gentamycin. Of the 3 quinolones tested, all strains of E. cloacae and C. freundii isolated were fully resistant to Norfloxacin and of Ofloxacin. For the 3 Sulfonamides+trimethoprim, all strains of C. freundii and S. typhi isolated were resistant to Nurofurantoin and Fosfomycin. Most of bacteria strains isolated in wastewater and surface water were resistant to all the antibiotics tested. Many bacterial strains tested were multi-resistant. This multidrug resistance was more marked in E. cloacae and C. freundii species isolated from wastewater and surface water. This represents a health risk for humans and the aquatic environment.